Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB.Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site.In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Simulation of protein crystal nucleation

To attempt to understand the physical principles underlying protein crystallization, an algorithm is described for simulating the crystal nucleation event computationally. The validity of the approach is supported by its ability to reproduce closely the well-known preference of proteins for particular space group symmetries. The success of the algorithm supports a recent argument that protein crystallization is limited primarily by the entropic effects of geometric restrictions imposed during nucleation, rather than particular energetic factors. These simulations provide a new tool for attacking the problem of protein crystallization by allowing quantitative evaluation of new ideas such as the use of racemic protein mixtures. Proteins 28:515–521, 1997. © 1997 Wiley-Liss, Inc.     

Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations

Myoglobin (Mb), the main cytosolic oxygen storage/deliver protein, is also known to interact with different small ligands exerting other fundamental physiological roles. In Humans up to five different Mb isoforms are present. The two most expressed ones (>90%) differ only at the 54th position, K54 (Mb-I) and E54 (Mb-II) respectively. High-altitude populations are characterized by a higher Mb concentration in skeletal muscle, totally attributable to Mb-II, as well as a higher efficiency of locomotion, leading to the hypothesis of a cause-effect relationship with the evolutionary response to the high-altitude hypoxic environment. In this work, a first structural characterization of the two more expressed human Mb isoforms has been carried out. In particular, a detailed (1)H and (129)Xe NMR study was aimed to characterize the structure of the hydrophobic cavities around the heme group. Experimental results have been compared to those from MD simulations, i.e. volume fluctuations and occurrence. Electronic structure of the heme ring ground state resulted to be comparable for the two investigated isoforms, despite the single point mutation at position 54. However, the use of (129)Xe as a probe revealed small but significant modifications in the structure of internal cavities. MD simulations supported NMR results indicating interesting structural/dynamical differences in the average volume and occurrence of the main cavities lining Mb prosthetic group.     

A Novel Injection-Molded Capsular Device for Oral Pulsatile Delivery Based on Swellable/Erodible Polymers

The feasibility of injection molding was explored in the preparation of a novel capsular device for oral pulsatile/delayed delivery based on swellable/erodible polymers. For this purpose, a mold intended to be coupled with a bench-top injection-molding press was designed. This was expected to enable the preparation of matching capsule cap and body items within a single manufacturing cycle and the selection of differing shell thicknesses (300, 600, and 900 μm). Hydroxypropylcellulose (Klucel^® EF, LF, and GF) was employed as the release-controlling polymer in admixture with polyethylene glycol 1500 (10%, w/w) as the plasticizer. After preliminary trials aimed at the setup of operating conditions, Klucel^® EF and LF capsule shells with satisfactory technological properties were manufactured. The performance of capsular devices filled with a tracer drug powder was studied by means of a modified USP31 disintegration apparatus. Typical in vitro delayed release patterns were thereby obtained, with lag time increasing as a function of the wall thickness. A good correlation was found between the latter parameter and t _10%, i.e., the time to 10% release, for both polymer grades employed. On the basis of the overall results, the investigated technique was proven suitable for the manufacturing of an innovative pulsatile release platform.     

Multimodal fMRI Resting-State Functional Connectivity in Granulin Mutations: The Case of Fronto-Parietal Dementia

Background

Monogenic dementias represent a great opportunity to trace disease progression from preclinical to symptomatic stages. Frontotemporal Dementia related to Granulin (GRN) mutations presents a specific framework of brain damage, involving fronto-temporal regions and long inter-hemispheric white matter bundles. Multimodal resting-state functional MRI (rs-fMRI) is a promising tool to carefully describe disease signature from the earliest disease phase.

Objective

To define local connectivity alterations in GRN related pathology moving from the presymptomatic (asymptomatic GRN mutation carriers) to the clinical phase of the disease (GRN- related Frontotemporal Dementia).

Methods

Thirty-one GRN Thr272fs mutation carriers (14 patients with Frontotemporal Dementia and 17 asymptomatic carriers) and 38 healthy controls were recruited. Local connectivity measures (Regional Homogeneity (ReHo), Fractional Amplitude of Low Frequency Fluctuation (fALFF) and Degree Centrality (DC)) were computed, considering age and gender as nuisance variables as well as the influence of voxel-level gray matter atrophy.

Results

Asymptomatic GRN carriers had selective reduced ReHo in the left parietal region and increased ReHo in frontal regions compared to healthy controls. Considering Frontotemporal Dementia patients, all measures (ReHo, fALFF and DC) were reduced in inferior parietal, frontal lobes and posterior cingulate cortex. Considering GRN mutation carriers, an inverse correlation with age in the posterior cingulate cortex, inferior parietal lobule and orbitofrontal cortex was found.

Conclusions

GRN pathology is characterized by functional brain network alterations even decades before the clinical onset; they involve the parietal region primarily and then spread to the anterior regions of the brain, supporting the concept of molecular nexopathies.     

Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.     

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.     

Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8(+) T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.     

Contrasting behavior of higher plant photosystem I and II antenna systems during acclimation

In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I.